Production of Haploid Plant of 'Banpeiyu'pummelo [Citrus Maxima

J. Japan. Soc. Hort. Sci. 79 (3): 239–245. 2010. Available online at www.jstage.jst.go.jp/browse/jjshs1 JSHS © 2010 Production of Haploid Plant of ‘Banpeiyu’ Pummelo [Citrus maxima (Burm.) Merr.] by Pollination with Soft X-Ray-Irradiated Pollen Masaki Yahata1**, Kiichi Yasuda1, Kohji Nagasawa2, Seiichi Harusaki2, Haruki Komatsu2 and Hisato Kunitake1* 1Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan 2School of Agriculture, Tokai University, Kumamoto 869-1404, Japan To induce haploid plants in Citrus maxima (Burm.) Merr. ‘Banpeiyu’, we evaluated the effect of pollination with soft X-ray-irradiated pollen on fruit set and seed development, and carried out ovule culture. When ‘Banpeiyu’ pummelo pistils were pollinated with X-ray-irradiated pollen of ‘Fukuhara’ sweet orange [C. sinensis (L.) Osbeck], the exposure doses affected the fruit set. The number of seeds per fruit was also affected by the exposure dose, and tended to decrease as the dose increased; however, all developed seeds obtained from these crosses were diploid. In the ovule culture of ‘Banpeiyu’ pummelo, six embryoids shown haploidy were obtained in all treatments. One haploid plantlet with 9 chromosomes was regenerated from an embryoid in a culture of ovules established 40 days after pollination with 400 Gray (Gy)-irradiated pollen of ‘Tosa-buntan’ pummelo (C. maxima). This haploid was suggested to be derived from ‘Banpeiyu’ pummelo by random amplified polymorphic DNA (RAPD) and cleaved amplified polymorphic sequence (CAPS) analysis. Key Words: chromosome observation, cleaved amplified polymorphic sequence (CAPS), flow cytometry, ovule culture, random amplified polymorphic DNA (RAPD). Introduction hybridization (Germanà and Chiancone, 2001; Oiyama and Kobayashi, 1993; Toolapong et al., 1996); however, Haploids have significant potential in Citrus breeding, these alternative methods have not been effective, and because homozygous plants, which are very important so it was difficult to carry out genetic analysis and for genetic analysis and breeding, are easily obtained by breeding using haploids. doubling their chromosomes. Through the fusion of Recent biological studies of various plants suggest haploid protoplasts, new forms of interspecific and that parthenogenesis induced by the pollination of intergeneric hybrids can be obtained (Grosser et al., inactivated pollen with or without a pseudo-fertilized 2000). Furthermore, gene transfer or mutagenesis of ovule culture might represent an alternative technique haploids, followed by chromosome doubling, would for the production of haploid plants in plant species in enable recessive genes to be expressed. In Citrus, haploid which anther culture is inefficient. Sauton and Dumas seedlings were first obtained by the application of γ-rays (1987) first succeeded in producing haploid plants in in natsudaidai (C. natsudaidai Hayata) (Karasawa, several cultivars of melon (Cucumis melo L.) using the 1971). Esen and Soost (1971) described a haploid pseudo-fertilized ovule culture technique. They cultured embryo obtained from an immature seed of clementine immature ovules after fertilization with pollen irradiated mandarin (C. clementina hort. ex Tanaka) × ‘Pearl’ with 300 Gy of γ-rays from a cobalt 60 source. Katoh tangelo cross. Since then, haploids have been produced et al. (1993) also reported high-frequency haploid by anther culture (Germanà and Chiancone, 2003; production in melon by the same pseudo-fertilized ovule Germanà et al., 1994; Hidaka et al., 1979) and interploid culture (maximum 3.4% per examined ovule), although the frequency varied with the genotype. In fruit trees, haploid plants induced by irradiated pollen have been Received; June 24, 2009. Accepted; January 15, 2010. * Corresponding author (E-mail: [email protected]). reported for apple (Malus domestica Borkh.) (Zhang and ** Present address: Faculty of Agriculture, Shizuoka University, Lespinasse, 1991), pear (Pyrus communis L.) (Bouvier Shizuoka 422-8529, Japan. et al., 1993), and kiwifruit [Actinidia deliciosa (A. Chev.) 239 240 M. Yahata, K. Yasuda, K. Nagasawa, S. Harusaki, H. Komatsu and H. Kunitake C. F. Liang & A. R. Ferguson] (Chalak and Legave, recorded. The seeds were extracted from each fruit and 1997; Pandey et al., 1990). classified into three groups, namely developed (normal In Citrus, on the other hand, there is only scant development), undeveloped (poor growth embryo), and information on parthenogenesis induced by inactivated other (empty and ovulelike) seeds. After all developed pollen (Froelicher et al., 2007; Ollitraut et al., 1996). seeds had been surface-dried and weighed and their seed Froelicher et al. (2007) produced five haploid plantlets coats had been removed, they were placed on double from three mandarin genotypes by pollinating with layers of moistened filter paper and kept at 25°C for pollen of Meyer lemon (C. meyeri Y. Tanaka) irradiated seven days. After germination, the seedlings were at 150 and 300 Gy of γ-rays from a cobalt 60 source. transplanted into vermiculite in boxes and transferred to In the present study, we describe in detail the effects a greenhouse. Undeveloped seeds were aseptically of pollination with soft X-ray-irradiated pollen on fruit cultured on Murashige and Skoog (MS) medium (1962) set and seed development, and demonstrate that containing 500 mg·L−1 malt extract, 30 g·L−1 sucrose, and pollination with irradiated pollen followed by ovule 2g·L−1 gellan gum without the seed coats being removed. culture in C. maxima was successful as a means of These cultures were kept at 25°C under continuous haploid production. illumination (38 μmol·m−2·s−1). Materials and Methods Ovule culture Plant materials Approximately 15 young fruits of ‘Banpeiyu’ ‘Banpeiyu’ pummelo, a monoembryonic diploid pummelo were collected 10, 20, and 40 days after cultivar, cultivated at the Fruit Research Institute, pollination with the 400 Gy-irradiated pollen of Kumamoto Prefectural Agricultural Research Center, ‘Fukuhara’ sweet orange and ‘Tosa-buntan’ pummelo. Matsubase, Kumamoto, Japan, was used as the seed ‘Banpeiyu’ pummelo fruits pollinated with non- parent. As pollen parents, ‘Fukuhara’ sweet orange and irradiated pollen of ‘Fukuhara’ sweet orange were used ‘Tosa-buntan’ pummelo, cultivated at the Citrus as controls. Ovules were excised aseptically from young Experimental Orchard, Faculty of Agriculture, Saga fruits with the aid of a dissecting microscope. The ovules University, were used. were cultured on Murashige and Tucker (MT) medium (1969) containing 500 mg·L−1 malt extract, 20 mg·L−1 Pollen irradiation adenine, 30 g·L−1 sucrose, and 2 g·L−1 gellan gum, and Collected pollen was placed on paraffin paper in a kept at 25°C under continuous illumination glass Petri dish (95 × 15 mm), which was transferred to (38 μmol·m−2·s−1). Callus, embryoid, and plant formation a soft X-ray unit (SV-100AW/I; Softex Co. Ltd., from these ovules was recorded 3 months after culture. Kanagawa, Japan) and irradiated with 0, 200, 400, 600, Plantlets obtained from the ovule culture were micro- 800, and 1000 Gy. grafted onto 20-day-old seedlings of trifoliate orange [Poncirus trifoliata (L.) Raf.]. Grafted plantlets were Pollen stainability and in vitro germination of irradiated directly transferred to sterilized soil in pots and grown pollen in a greenhouse, which was air-conditioned to maintain ‘Fukuhara’ sweet orange was used for testing pollen a temperature of about 20°C. stainability and in vitro germination of irradiated pollen. Pollen stainability was estimated with acetocarmine Ploidy level analysis crushing of nearly mature anthers. In vitro germination Flow cyotometry of pollen grains was performed on a glass slide covered Tissue segments were collected from the embryoids with a 2 mm layer of 1% (w/v) agar medium containing or developing leaves of a plantlet, chopped with a razor 10% sucrose. Five dehisced anthers, each from different blade in 2 mL buffer solution containing 1.0% (v/v) flowers, were rubbed on the agar medium, and the slides Triton X-100, 140 mM mercaptoethanol, 50 mM were then incubated for 10 h in a moistened chamber at Na2SO3, and 50 mM Tris-HCl at pH 7.5, and incubated 25°C in the dark. For each irradiation dose, pollen for 5 min according to the preparation method of Yahata stainability and in vitro germination rates were evaluated et al. (2005a). Crude samples were filtered through from three hundred grains with three repetitions. Miracloth (Merck KGaA, Darmstadt, Germany) and stained with 25 μg·L−1 propidium iodide (PI). The Effect of pollination with soft X-ray-irradiated pollen on relative fluorescence of total DNA was measured for fruit and seed development each nucleus with a Flow Cytometry System (EPICS Twenty-five flowers of ‘Banpeiyu’ pummelo were XL; Beckman Coulter, Fullerton, CA, USA) equipped hand-pollinated with pollen of ‘Fukuhara’ sweet orange with an argon laser (488 nm, 15 mW). irradiated at each exposure rate, and were immediately Chromosome observation covered with paraffin paper bags after emasculation. Young leaves (approximately 3–5 mm long) were Fruits were harvested at maturity between November excised from the plantlet, immersed in 2 mM 8- and early January, and fruit sets and weights were hydroxyquinoline for 6 h at 4°C, and fixed in a mixed J. Japan. Soc. Hort. Sci. 79 (3): 239–245. 2010. 241 solution of ethanol and acetic acid (3 : 1) for 12 h at 4°C. 11.8% in vitro germination (Table 1). There were no Enzymatic maceration and air drying were performed significant differences in stainability and in vitro according to the method of Fukui (1996) with some germination rates among the different doses of soft X- modifications. Young leaves were washed in distilled ray irradiation. It has been shown that irradiation of water to remove the fixative and then macerated in an pollen hardly affects stainability or in vitro germination enzyme mixture containing 2% (w/v) Cellulase Onozuka in apple (Zhang and Lespinasse, 1991), melon (Sauton RS (Yakult Pharmaceutical Ind. Co. Ltd., Tokyo, Japan), and Dumas, 1987), kiwifruit (Musial and Przywara, 1% (w/v) Macerozyme R-200 (Yakult Pharmaceutical 1998) and cacao (Theobroma cacao L.) (Falque et al., Ind.

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